The central goal of the experiments in this proposal is to understand how neurons find their targets during development. It is now clear that this process involves the combined actions of attractive and repulsive guidance cues, both of which can act at short and long distances to steer neuronal growth cones. Recent work on members of the phylogenetically conserved semaphorin gene family, many of which can function in vivo and in vitro as repellents, suggests the exciting possibility that a thorough understanding of the cellular and molecular basis of semaphorin function will yield general principles of repulsive guidance mechanisms. Understanding how semaphorins affect the neuronal growth cone cytoskeleton will assist in understanding repulsive and inhibitory events that occur during adult life following neuronal injury or neuronal degeneration. In addition, since semaphorins function in immune system signaling events and have also been linked to the progression of certain types of cancers, the experiments we propose will have implications that extend far beyond how semaphorins mediate axon guidance during neural development. We have shown that the transmembrane semaphorin Sema 1a, like secreted semaphorins, functions in vivo to mediate repulsive guidance events. This work has resulted in the development of several genetic and molecular paradigms designed to identify the downstream signaling components used by Sema 1a to mediate repulsive neuronal guidance events. We have also begun an analysis of the function of certain vertebrate transmembrane semaphorins during neural development and have identified a novel protein (TSBP) that binds with high affinity to vertebrate thrombospondin domain-containing semaphorins and will aid in this endeavor. Accordingly, we propose to identify downstream signaling components that transduce repulsive neuronal guidance events mediated by Sema 1a in Drosophila using genetic and molecular strategies. We will also determine the role in vertebrate neural development played by thrombospondin domain-containing transmembrane semaphorins, using functional assays in defined embryonic neuronal populations and undertaking a detailed cellular and molecular analysis of TSBP.